The long-term goal of this research program is to develop an efficient large-scale commercial system for manufacture of lentiviral gene therapy vectors in suspension cells. This proposal is submitted in response to PA-06-013, "Manufacturing Processes of Medical, Dental and Biological Technologies." There is a critical unmet need to develop robust, scalable, closed, cGMP-compliant manufacturing systems for generating and producing lentivirus gene therapy vectors that are suitable for clinical and commercial use. Lentiviral vectors are being developed for a number of therapeutic applications and clinical studies are underway. Unfortunately, lentiviral vectors are difficult to produce in large numbers due to the lack of stable packaging cell lines and due to inefficiencies, lack of robustness, and limited scalabilities associated with standard methods using transient transfection, which is a key step in the manufacturing process. MaxCyte has developed a cell transfection and production system that is an efficient, scalable process for engineering cell function and enabling cell-based manufacturing for clinical applications. In Phase 1 of this project, we demonstrated that this system enables high titer lentivector production in adherent and suspension-adapted cells in both small and large scale transfections. The next challenge is to optimize and scale up an aseptic production system and demonstrate large scale lentivector production technology in a cGMP manufacturing facility. This achievement will enable the first application of an FDA-approved cell loading technology for scalable production of lentiviral vectors by transient transfection in non-adherent cells cultured in serum-free medium. Therefore, the Specific Aims of this Phase 2 project are to: 1) optimize culture conditions for culturing suspension-adapted cells for vector production;2) achieve sterile integration of the MaxCyte cell processing instrumentation with the Wave cell production technology;3) optimize viral production capacity by maximizing the number of infectious viral particles that can be produced by and collected from transfected cells;4) demonstrate feasibility of the completely integrated viral production system at a small commercial scale;and 5) demonstrate large scale lentivector production in a cGMP manufacturing facility. The interest expressed in this application of the MaxCyte cell loading technology by Indiana University Vector Production Facility (IUVPF) and SAFC Pharma emphasizes the importance of the project. These facilities provide contract manufacturing services for lentiviral vectors (and a number of other viral vectors) for gene therapy clinical trials. IUVPF will perform cGMP manufacturing for Aim 5 and thereby demonstrate the ability to transfer the MaxCyte technology to large scale commercial manufacturing. Public Health Relevance: There is a critical unmet need to develop robust, scalable, closed, cGMP-compliant manufacturing systems for producing lentiviral gene therapy vectors that are suitable for clinical and commercial use. The goal of this Phase 2 SBIR project is to optimize and validate a proprietary cell transfection and production system that provides an efficient, large-scale commercial process for developing and manufacturing lentiviral gene therapy vectors for clinical applications. The benefits of this production system will translate into safer, better, cost effective gene therapies on the market more rapidly to benefit patients in many disease areas with unmet need today, such as: Parkinson's disease, age-related macular degeneration, beta-thalassemia, sickle-cell anemia, muscular dystrophy, and a variety of lympho-hematopoetic pathologies including HIV infection, leukemias, and lymphomas.